Fluorescent compound, fluorescent compound mixture, freshness marker, freshness label, and sensing system

ABSTRACT

Disclosed herein is a fluorescent compound represented by the following general formula and having fluorescence characteristics that vary in the presence of a subject substance, 
                         
wherein R 1  and R 2  each independently represent a carboxyl group, or a substituent containing a carboxyl group, and R 3  and R 4  each independently represent a hydrogen atom, an alkyl group, an alkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or a substituent containing a carboxyl group.

CROSS-REFERENCE TO RELATED APPLICATION

This application is based upon and claims the benefit of priority fromJapanese Patent Application 2017-011618 filed on Jan. 25, 2017, theentire contents of which are incorporated herein by reference.

FIELD

Embodiments described herein relate generally to a fluorescent compound,a fluorescent compound mixture, a freshness marker, a freshness label,and a sensing system.

BACKGROUND

Amines generated by food spoilage (hereinafter, referred to as “biogenicamines”, or, simply, “amines”) in aquatic and meat products pose healthrisks such as allergic disease and food poisoning. Biogenic amines areproduced by decarboxylation reaction of amino acid, and generate alsoduring processing or storage of food. The amount of generated biogenicamine can thus be used as an index of freshness in food. For theprevention of allergic disease and food poisoning, and the reduction offood waste, there is a need for a method for sensing biogenic amine withhigh sensitivity and high selectivity.

Known biogenic amine sensing methods use analytical instruments such asgas chromatography and liquid chromatography devices. However, sensingmethods using such analytical instruments take time because thesemethods require processing before measurement, and the devices need tobe constantly monitored for adjustment. These add to the cost of thesensing methods using analytical instruments.

A method using a tetraphenylethene fluorescent compound is proposed asan easy and quick biogenic amine sensing method. This sensing methoduses a solution of tetraphenylethene fluorescent compound. Dissolving abiogenic amine in the solution causes the tetraphenylethene fluorescentcompound to react with the biogenic amine, and form an aggregate in thesolution. The tetraphenylethene fluorescent compound produces only aweak fluorescence intensity by itself, but is known to produce astronger fluorescence intensity upon forming an aggregate. An increasedfluorescence intensity can be detected as an indication of biogenicamine generation. The detection result can then be determined as sensingof deterioration of freshness in food. However, the sensitivity tobiogenic amine is still insufficient.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram representing an example of a biogenic amine sensingprocess using a freshness marker according to an exemplary embodiment.

FIG. 1B is a diagram representing an example of a biogenic amine sensingprocess using a freshness marker according to an exemplary embodiment.

FIG. 2A is a diagram representing an example of a biogenic amine sensingprocess using a freshness label according to an exemplary embodiment.

FIG. 2B is a diagram representing an example of a biogenic amine sensingprocess using a freshness label according to an exemplary embodiment.

FIG. 3 is a graph representing an example of a fluorescence intensitychange before and after formation of an aggregate by a freshness markeraccording to an exemplary embodiment.

FIG. 4 is a diagram representing the relationship between fluorescenceintensity and the fraction of cis-fluorescent compounds in a fluorescentcompound of an Example.

FIG. 5 is a diagram representing the relationship between wavelength andthe fluorescence intensity of a fluorescent compound of an Example.

FIG. 6 is a diagram representing the fluorescence intensity of thefluorescence emitted by a fluorescent compound according to an exemplaryembodiment after addition of spermidine.

FIG. 7A is a diagram explaining a fabrication method of a freshnesslabel according to an exemplary embodiment.

FIG. 7B is a diagram explaining a fabrication method of a freshnesslabel according to an exemplary embodiment.

FIG. 7C is a diagram explaining a fabrication method of a freshnesslabel according to an exemplary embodiment.

FIG. 8 is a diagram explaining an evaluation method for a freshnesslabel according to an exemplary embodiment.

FIG. 9 is an image showing fluorescence states of freshness labelsaccording to an exemplary embodiment.

DETAILED DESCRIPTION

A fluorescent compound according to an embodiment is represented by thefollowing general formula (1), and has fluorescence characteristics thatvary in the presence of a subject substance.

In the formula, R₁ and R₂ each independently represent a carboxyl group,or a substituent containing a carboxyl group. R₃ and R₄ eachindependently represent a hydrogen atom, an alkyl group, an alkoxygroup, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group.

The embodiment is described below in detail with reference to theaccompanying drawings. The embodiment is based on a fluorescent compoundthat is either a cis-tetraarylethene derivative or atrans-tetraarylethene derivative. The tetraarylethene derivativeaggregates, and its fluorescence characteristics vary in the presence ofan amine. The fluorescent compound according to the embodiment undergoesa larger fluorescence intensity change upon aggregation than atetraarylethene derivative containing a cis-tetraarylethene derivativeand a trans-tetraarylethene derivative in the same fraction. This makesit easier to observe a fluorescence intensity upon aggregation.

Biogenic Amine

If left unchecked, food typically undergoes changes in quality such assmell, appearance, texture, and taste over time before it is no longersuited for consumption. Such degeneration of food condition is calleddeterioration, decaying, or degradation, or, more commonly, “foodspoilage”. Food deterioration is caused by microorganisms, insects,self-digestion, chemical (lipid oxidation, browning), or physical(damage such as cuts and crushes) causes. In many cases, fooddeterioration is caused by proliferation of microorganisms (putrefactivebacteria). In a broad sense, the term “spoilage” is used to describesuch deterioration of food by proliferation of microorganisms to thepoint where it is no longer edible.

Spoilage refers to the process by which protein in food decomposes bythe effects of microorganisms, and produces harmful substances or a badodor. This is often distinguished from “decaying”, which describes astate in which carbohydrates and fats decompose by the effects ofmicroorganisms, and produce a bad flavor not suitable for consumption.The term “degradation” describes a state where such spoilage or decayingmakes the food not suitable for consumption. The main components of afoul odor are various amine components also called biogenic amines suchas ammonia, and trimethylamine.

The nitrogen compounds in food are mainly proteins, which becomehydrolyzed by the enzymes of microorganisms and food into polypeptides,and to simple peptides or amino acids. These amino acids decomposethrough reactions such as deamination, transamination, anddecarboxylation, and produce biogenic amines.

Examples of the biogenic amines produced by amino acids include1,2-ethylenediamine, 1,3-propanediamine, 1,4-butanediamine,1,5-pentanediamine, 1,6-hexanediamine, spermidine, spermine, histamine,and tryptamine.

Fluorescent Compound

The fluorescent compound according to the embodiment aggregates, orprecipitates into crystals in the presence of an amine, and changes itsfluorescence characteristics, including fluorescence or excitationspectrum characteristics, and fluorescence lifetime. The fluorescentcompound emits weak fluorescence when exposed to excitation light in adissolved state in a solvent. The fluorescence intensity increases asthe fluorescent compound according to the embodiment aggregates in thesolvent.

Specifically, the fluorescent compound according to the embodiment is asimple tetraarylethene derivative represented by the following generalformula (1) or (2), or a mixture of tetraarylethene derivativesrepresented by the following general formulae (1) and (2).

In the general formula (1), R₁ and R₂ each independently represent acarboxyl group, or a substituent containing a carboxyl group. R₃ and R₄each independently represent a hydrogen atom, an alkyl group, an alkoxygroup, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group.

In the general formula (2), R₅ and R₈ each independently represent acarboxyl group, or a substituent containing a carboxyl group. R₆ and R₇each independently represent a hydrogen atom, an alkyl group, an alkoxygroup, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group.

The tetraarylethene derivatives represented by the foregoing formulae(1) and (2) are synthesized from a compound represented by the followinggeneral formula (3), using a known reaction technique. For synthesis ofthe tetraarylethene derivative, the compounds represented by thefollowing general formula (3) may be used as a mixture of two or more,as required.

In the general formula (3), R₉ represents a carboxyl group, or asubstituent containing a carboxyl group. R₁₀ represents a hydrogen atom,an alkyl group, an alkoxy group, a halogen atom, a hydroxyl group, acarboxyl group, or a substituent containing a carboxyl group.

In the embodiment, non-limiting examples of the substituent containing acarboxyl group include —(CH₂)_(m)—COOH, —O—(CH₂)_(m)—COOH,—S—(CH₂)_(m)—COOH, —NH—(CH₂)_(m)—COOH, —O—(CH₂)_(m)—S—(CH₂)_(n)—COOH,—O—(CH₂)_(m)—NH—(CH₂)_(n)—COOH, —S—(CH₂)_(m)—O—(CH₂)_(m)—COOH,—S—(CH₂)_(m)—NH—(CH₂)_(n)—COOH, —NH—(CH₂)_(m)—O—(CH₂)_(n)—COOH, and—NH—(CH₂)_(m)—S—(CH₂)_(n)—COOH (where m and n represent an integer of 1to 6).

In the embodiment, non-limiting examples of the alkyl group includelinear or branched alkyl groups of 1 to 6 carbon atoms, for example,such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl,t-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl,n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl,1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl,1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl,and 2-ethylbutyl.

In the embodiment, non-limiting examples of the alkoxy include linear orbranched alkoxy of 1 to 6 carbon atoms, for example, such as methoxy,ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy,sec-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy, n-hexyloxy,isohexyloxy, and 3-methylpentyloxy.

When R₉ and R₁₀ in the compound represented by the general formula (3)are different substituents, the synthesized tetraarylethene derivativehas cis and trans isomers (geometric isomers). The tetraarylethenederivative is a mixture of the tetraarylethene derivatives representedby the general formulae (1) and (2), and the tetraarylethene derivativesrepresented by the general formulae (1) and (2) are cis and transisomers.

In the mixture of tetraarylethene derivatives, the tetraarylethenederivative represented by the general formula (1) is a cis-fluorescentcompound, and the tetraarylethene derivative represented by the generalformula (2) is a trans-fluorescent compound.

As a rule, a cis-form has two substituents on the same side of thedouble bond, whereas a trans form has two substituents on the oppositesides of the double bond. As used herein, “cis-fluorescent compound”refers to a fluorescent compound that has a carboxyl group, or asubstituent containing a carboxyl group disposed on the adjacent benzenerings. On the other hand, “trans-fluorescent compound” as used hereinrefers to a fluorescent compound that has a carboxyl group, or asubstituent containing a carboxyl group on benzene rings that are onopposite sides of the double bond.

As a rule, cis and trans isomers are obtained as an equimolar mixture ofcis and trans isomers. Accordingly, the tetraarylethene derivativemixture obtained after the synthesis contains the same number of molesof the cis-fluorescent compound and the trans-fluorescent compoundrepresented by the general formulae (1) and (2), respectively. Byseparation and purification of the tetraarylethene derivative mixture, atetraarylethene derivative of primarily the cis-fluorescent compoundrepresented by the general formula (1), or a tetraarylethene derivativeof primarily the trans-fluorescent compound represented by the generalformula (2) can be obtained.

The tetraarylethene derivatives represented by the general formulae (1)and (2) produce weak fluorescence in a dissolved state in a solvent. Inthe presence of an amine, the tetraarylethene derivative aggregates asit becomes less soluble in solution through hydrogen bonding orelectrostatic interaction (hereinafter, also referred to as “reaction”)of the carboxyl group in the molecule with the amine. The aggregatedtetraarylethene derivative emits fluorescence of higher fluorescenceintensity when exposed to excitation light such as ultraviolet (UV)light. This change in the fluorescence characteristics occurs as theaggregation of the tetraarylethene derivative restricts the arylrotation in the tetraarylethene derivative. In the embodiment, thefluorescent compound is dissolved in a solvent in such a concentrationthat the unreacted fluorescent compound does not aggregate orprecipitate, or does not become saturated.

A tetraarylethene derivative solution prepared by dissolving thetetraarylethene derivative in a solvent produces fluorescence of highfluorescence intensity in the presence of an amine. Observed changes inthe fluorescence intensity of the fluorescence produced by the solutioncan thus be detected as an indication of the presence of an amine.

Freshness Marker

The fluorescent compound according to the embodiment may be used for afreshness marker. The freshness marker includes the fluorescent compoundaccording to the embodiment, and a solvent dissolving the fluorescentcompound according to the embodiment.

Solvent

The solvent according to the embodiment is a solvent capable ofdissolving the fluorescent compound, and having good compatibility withthe biogenic amine to be sensed. Use of a solvent that is poorlycompatible with the biogenic amine may result in formation ofheterogeneous aggregates, and cause poor freshness marker sensitivity.

Because the freshness marker is used to sense deterioration of food overtime, the solvent according to the embodiment is preferably a solventthat does not undergo evaporative volume reductions in the atmosphereover a certain length of time. Further, because the freshness marker isused by being attached to food or being installed in the vicinity offood, the solvent according to the embodiment is preferably a solventthat is safe to the human body. The solvent according to the embodimentmay be a mixed solvent of two or more solvents.

Preferred as such a solvent is a glycol-based solvent having a highboiling point and low toxicity. Specific examples of such glycol-basedsolvents include ethylene glycol-based solvents such as polyethyleneglycol monomethyl ether, diethylene glycol ethyl methyl ether,polyethylene glycol dimethyl ether, triethylene glycol butyl methylether, and diethylene glycol butyl methyl ether; and propyleneglycol-based solvents such as propylene glycol monomethyl ether,propylene glycol monobutyl ether, and propylene glycol dimethyl ether.

The freshness marker according to the embodiment may be used as, forexample, a spoiled substance detection chemical solution for detecting aspoiled substance.

FIGS. 1A and 1B are diagrams representing an example of a biogenic aminesensing process using the freshness marker according to the embodiment.As illustrated in FIG. 1A, a freshness marker 1 according to theembodiment includes a fluorescent compound 10, and a solvent 11dissolving the fluorescent compound 10. The freshness marker 1 accordingto the embodiment is contained in a glass container G. As illustrated inFIG. 1B, a biogenic amine 12 a in biogenic amines generated from a foodproduct F has good compatibility with the solvent 11, and forms anaggregate 13 with the fluorescent compound 10 dissolved in the solvent11. The freshness of the food product F can be determined by irradiatingexcitation light such as UV light to the freshness marker 1 from outsideof the glass container G.

The freshness marker 1 has use for, for example, a spot test in a foodfactory. The freshness of the subject food product F can be determinedby immersing a portion of the food product F in the freshness marker 1contained in the glass container G. The food product F being testedcontacts the freshness marker 1, and the fluorescent compound 10 in thefreshness marker 1 quickly forms the aggregate 13 by reacting with abiogenic amine.

The freshness of the food product F also can be determined withoutimmersing the food product F in the freshness marker 1. The freshnessmarker 1 may be used by being added to the food product F, or by beinginstalled in the vicinity of the food product F. In the case of thefreshness marker 1 installed in the vicinity of the food product F, thefluorescent compound reacts with biogenic amine vapor upon generation ofbiogenic amine from a spoiled food product F, and emits high-intensityfluorescence. A change in fluorescence intensity can be detected as anindication of biogenic amine generation to determine the freshness ofthe food product F.

Freshness Label

The freshness marker 1 can be used to provide a freshness label. FIGS.2A and 2B are diagrams representing an example of a biogenic aminesensing process using the freshness label according to the embodiment. Afreshness label 100 is used for the detection of a biogenic aminegenerated from a food product F.

As illustrated in FIG. 2A, the freshness label 100 according to theembodiment includes medium 14 impregnated with the freshness marker 1that includes the fluorescent compound 10 and the solvent 11.

Medium

The medium 14 according to the embodiment can retain the freshnessmarker 1. Considering retention of the freshness marker 1, the medium 14is preferably one with a certain level of porosity, for example, aporous substrate, or a mesh structure.

Examples of such medium 14 include a cellulose fiber, paper, a fabric, afilter, and a sponge. Particularly preferred is a cellulose acetatemembrane filter because it can enhance fluorescence intensity.

Preferably, the medium 14 is selected from materials having refractiveindices as close as possible to the refractive index of the solvent 11of the embodiment. With the medium 14 and the solvent 11 having similarrefractive indices, the fluorescence generated inside the medium 14 willnot be blocked. This makes it possible to obtain even higherfluorescence intensity, and the freshness marker 1 using the medium 14is advantageous for freshness determination.

The freshness label is disposed near the food product F, for example. Asillustrated in FIG. 2B, the food product F generates biogenic amines 12a and 12 b, and the biogenic amine 12 a having good compatibility withthe solvent 11 forms the aggregate 13 with the fluorescent compound 10retained in the medium 14. The freshness of the food product F can bedetermined by irradiating excitation light such as UV light to thefreshness label 100.

The food product F, which is a food material such as fish and meat, ishandled by, for example, being sealed (air tight) in a plasticcontainer. The freshness of the food product F can be determined fromoutside of the container by attaching the freshness label 100 inside thecontainer containing the food product F. The fluorescent compound 10aggregates as it reacts with the biogenic amine 12 a generated from thefood product F. The freshness of the food product F can be determined byirradiating excitation light such as UV light to the freshness label 100from outside of the container containing the food product F, withoutleaking air from the container.

Base Material

A base material that supports the freshness marker 1 or the freshnesslabel 100 may be used, as required. Preferably, the base material isselected from materials that are resistant to the solvent 11 dissolvingthe fluorescent compound 10, and that do not emit fluorescence bythemselves. However, the base material is not limited to such materials,and any material may be used with the provision that the fluorescencewavelength is not close to the fluorescence wavelength of thefluorescent compound 10 emitting fluorescence.

Examples of such base materials include plastic sheets such as a Teflon®sheet, a polyimide sheet, a polyester film, a polyacetal sheet, a nylonsheet, a polycarbonate sheet, a polypropylene sheet, a polyethylenesheet, a PET film, and a vinyl chloride sheet; and glass plates.

Determination Method

The freshness marker 1 according to the embodiment is used to determinethe freshness of a food product F by sensing changes in the fluorescencecharacteristics of the fluorescent compound 10, as described above. Thefreshness marker 1 according to the embodiment may be used for a sensingsystem for sensing a subject substance.

A sensing system according to the embodiment is configured from thefreshness marker according to the embodiment, a ultraviolet source unitthat irradiates UV light to the freshness marker, and an emissiondetector that detects an image pattern that occurs in the freshnessmarker upon irradiation of the freshness marker with UV light. Thesensing system according to the embodiment senses a subject substanceusing an image pattern that occurs in the freshness marker uponirradiation of the freshness marker with UV light, and enablesdetermination of the freshness of a food product. Here, the emissiondetector means an imaging device such as a digital camera.

Specifically, a food product F is placed in the freshness marker 1. Thefreshness marker 1 is then irradiated with UV light using theultraviolet source, and imaged with a digital camera. This can producean image of the freshness marker 1.

The fluorescent compound 10 of the freshness marker 1 that reacted withthe biogenic amine 12 a generated from the food product F shows afluorescence characteristics change. The freshness of the food product Fcan thus be determined from changes occurring in, for example, thecolor, and the luminance of the freshness marker 1 in the producedimage. The determination of the freshness of the food product F may bemade with a program that automatically determines, for example, thecolor, and the luminance of the freshness marker 1 appearing in theimage. The program may be provided in the emission detector.

The electronic image produced by a digital camera or other such devicesmay be processed to enhance contrast. Such image processing is effectivewhen a slight difference in fluorescence intensity, specifically, aslight difference in the amount of generated biogenic amine needs to bedistinguished. It is also possible to use, for example, a smartphonewith a camera having a colorimetric function, and automaticallydetermine a fluorescence intensity difference in the image fordetermination of the freshness of a food product F.

The freshness of a food product F also may be determined by visuallyinspecting the freshness marker 1. When determining the freshness of afood product F through visual inspection, it is desirable to observe thefreshness marker 1 in an environment where the influence of visiblelight is minimal, such as in a darkroom. The accuracy of freshnessdetermination can improve when a fluorescence photometer is used.

Fluorescence Characteristics Changes

The fluorescent compound 10 is synthesized from the compound representedby the general formula (3). When the substituents R₉ and R₁₀ of thecompound are different, the fluorescent compound is a mixture of acis-fluorescent compound and a trans-fluorescent compound. In thefluorescent compound 10 of the freshness marker 1, the cis-fluorescentcompound and the trans-fluorescent compound forms an assembly. Anassembly of cis-fluorescent compound and trans-fluorescent compoundfluoresces as the aryl rotation is restricted. This causes the freshnessmarker 1 to emit fluorescence even before forming the aggregate 13.

In the presence of the biogenic amine 12 a, the fluorescent compound 10fluoresces by forming the aggregate 13 through reaction with thebiogenic amine 12 a. That is, the freshness marker 1 also emitsfluorescence after forming the aggregate 13.

FIG. 3 is a graph representing an example of the fluorescence intensityof the freshness marker 1 before and after formation of the aggregate13. The fluorescent compound 10 of the freshness marker 1 is 95 mol %cis-fluorescent compound and 5 mol % trans-fluorescent compound. Themeasured characteristics curve c represents the relationship between thefluorescence intensity of the freshness marker 1 and wavelength afterthe formation of the aggregate 13 with a 6 molar equivalent ofspermidine added to the freshness marker 1. The measured characteristicscurve d represents the relationship between the fluorescence intensityof the freshness marker 1 and wavelength before formation of theaggregate 13. By comparing the characteristics curve c and thecharacteristics curve d, the fluorescence intensity represented by thecharacteristics curve c is higher than the fluorescence intensityrepresented by the characteristics curve d.

The fluorescence emitted by the freshness marker 1 becomes more clearlyobservable as the fluorescence intensity a of the fluorescence emittedby the freshness marker 1 after the formation of the aggregate 13increases. The freshness marker 1 emitting fluorescence of highfluorescence intensity a is therefore advantageous for freshnessdetermination. A fluorescence intensity change in the fluorescenceemitted by the freshness marker 1 also becomes more clearly observableas the ratio of fluorescence intensity a increases relative to thefluorescence intensity b of the fluorescence emitted by the freshnessmarker 1 before formation of the aggregate 13. The freshness marker 1emitting fluorescence with a larger ratio of fluorescence intensity arelative to fluorescence intensity b is therefore advantageous forfreshness determination.

FIG. 4 is a graph representing the relationship between fluorescenceintensity and the fraction of cis-fluorescent compounds in thefluorescent compound 10. In FIG. 4, the left vertical axis representsthe fluorescence intensity a of the fluorescence emitted by thefreshness marker 1 after the formation of the aggregate 13, and theright vertical axis represents the fluorescence intensity b of thefluorescence emitted by the freshness marker 1 before formation of theaggregate 13. As shown in FIG. 4, the fluorescence intensity b is thehighest, and the difference from the fluorescence intensity a is thesmallest when the fluorescent compound 10 is 50 mol % cis-fluorescentcompound and 50 mol % trans-fluorescent compound.

The assembly of cis-fluorescent compound and trans-fluorescent compoundcontains the same number of moles of cis-fluorescent compound andtrans-fluorescent compounds. Specifically, all the cis-fluorescentcompound and all the trans-fluorescent compound form the assembly in afluorescent compound that is 50 mol % cis-fluorescent compound and 50mol % trans-fluorescent compound. As a rule, the fluorescence intensityof the fluorescence emitted by a fluorescent compound dissolved in asolvent increases as the fluorescent compound concentration increases.Accordingly, the fluorescence intensity b of the fluorescence emitted bythe freshness marker 1 increases when all the cis-fluorescent compoundsand all the trans-fluorescent compounds form the assembly. This makesthe ratio of fluorescence intensity a smaller relative to thefluorescence intensity b, and makes the freshness determinationdifficult.

On the other hand, the fluorescent compound 10 containing a largerfraction of cis-fluorescent compounds or trans-fluorescent compoundscontains cis-fluorescent compounds or trans-fluorescent compounds thatdo not form the assembly, and accordingly the fraction of the assemblyformed by cis-fluorescent compounds and trans-fluorescent compoundbecomes smaller. The freshness marker 1 using such fluorescent compound10 has a smaller fluorescence intensity b, and the ratio of fluorescenceintensity a increases relative to the fluorescence intensity b. Thefreshness marker 1 using such fluorescent compound 10 is thereforeadvantageous for freshness determination.

For example, the fluorescent compound 10 synthesized from4-benzoylbenzoic acid is dissolved in a solvent to produce a 500 μM(molar concentration) solution, and a 6 molar equivalent of spermidineis added. When the fluorescent compound 10 is 50 mol % cis-fluorescentcompound and 50 mol % trans-fluorescent compound, the fluorescenceintensity a is higher than the fluorescence intensity b by a factor of48. On the other hand, when the fluorescent compound 10 is 15 mol %cis-fluorescent compound and 85 mol % trans-fluorescent compound, thefluorescence intensity a is higher than the fluorescence intensity b bya factor of 83. When the fluorescent compound 10 is 95 mol %cis-fluorescent compound and 5 mol % trans-fluorescent compound, thefluorescence intensity a is higher than the fluorescence intensity b bya factor of 86. As shown by these numbers, the freshness marker 1 usingthe fluorescent compound 10 having a larger fraction of cis-fluorescentcompounds or trans-fluorescent compounds has a larger ratio offluorescence intensity a relative to fluorescence intensity b, and isadvantageous for freshness determination.

The cis-fluorescent compound and the trans-fluorescent compound areformed by bonds represented by the following structural formulae (4) and(5), respectively, and the fluorescence characteristics are different.The structural formulae (4) and (5) show simplified structures withoutstructures such as the benzene ring.

The cis-fluorescent compound contains aryls that are adjacent to eachother, and the aryl rotation is restricted. This increases thefluorescence intensity of the emitted fluorescence. On the other hand,the aryls in the trans-fluorescent compound are on opposite sides of thedouble bond, and the aryl rotation is less restricted than in thecis-fluorescent compound. The fluorescence intensity of the emittedfluorescence is accordingly lower than in the cis-fluorescent compound.

FIG. 5 is a graph representing the relationship between wavelength andthe fluorescence intensities of fluorescent compounds according toExamples. The characteristics curve X represents the relationshipbetween wavelength and the fluorescence intensity of the fluorescentcompound 10 of primarily cis-fluorescent compound with a cis-fluorescentcompound fraction of 95 mol % and a trans-fluorescent compound fractionof 5 mol %. The characteristics curve Y represents the relationshipbetween wavelength and the fluorescence intensity of the fluorescentcompound 10 of primarily trans-fluorescent compounds with acis-fluorescent compound fraction of 15 mol % and a trans-fluorescentcompound fraction of 85 mol %. The characteristics curve Z representsthe relationship between wavelength and the fluorescence intensity ofthe fluorescent compound 10 that is 50 mol % cis-fluorescent compoundand 50 mol % trans-fluorescent compound.

By comparing the characteristics curves X and Y, the fluorescenceintensity represented by the characteristics curve X is higher than thefluorescence intensity represented by the characteristics curve Y. Thatis, the fluorescent compound 10 containing a larger fraction ofcis-fluorescent compounds than trans-fluorescent compounds produces ahigher fluorescence intensity a than the fluorescent compound 10containing a larger fraction of trans-fluorescent compounds thancis-fluorescent compounds.

When the fluorescence characteristics changes of the freshness marker 1are observable by visual inspection of the freshness marker 1, thefreshness can be determined by a simple method without using a digitalcamera or other devices. Fluorescent compounds 10 containing differentfractions of cis-fluorescent compounds were used to produce differentfreshness markers 1, and the freshness markers 1 were visually observedfor fluorescence emission. The visual inspection confirmed brighterfluorescence in the fluorescent compound 10 that contained twice as muchcis-fluorescent compound as trans-fluorescent compound than in thefluorescent compound 10 that was 50 mol % cis-fluorescent compound and50 mol % trans-fluorescent compound. The visual inspection alsoconfirmed much brighter fluorescence in the fluorescent compound 10 thatcontained cis-fluorescent compounds in three times the amount of thetrans-fluorescent compounds than in the fluorescent compound 10 that was50 mol % cis-fluorescent compound and 50 mol % trans-fluorescentcompound.

It is therefore preferable that the freshness marker 1 use thefluorescent compound 10 that contains a larger fraction ofcis-fluorescent compounds than trans-fluorescent compounds. Morepreferably, the fluorescent compound 10 contains at least twice as muchcis-fluorescent compound than trans-fluorescent compound. It is furtherpreferable to use the fluorescent compound 10 that contains thecis-fluorescent compound in at least three times the amount of thetrans-fluorescent compound.

The exemplary embodiment is described below in greater detail usingExamples and Comparative Examples. The following descriptions are not tobe construed as limiting.

Freshness markers of Examples 1 to 3 and Comparative Example 1 wereproduced using a fluorescent compound containing the cis-fluorescentcompound represented by the following general formula (6), and thetrans-fluorescent compound represented by the following general formula(7), and a fluorescent compound having two cis structures represented bythe following general formula (8). As used herein, “cis structure”refers to a structure in which the carboxyl groups, or substituentscontaining carboxyl groups are disposed on the adjacent benzene rings.

The cis-fluorescent compound represented by the general formula (6) isthe cis-fluorescent compound of general formula (1) of when R₁ and R₂are carboxyl groups, and R₃ and R₄ are hydrogen atoms. Thetrans-fluorescent compound represented by the general formula (7) is thetrans-fluorescent compound of general formula (2) of when R₅ and R₈ arecarboxyl groups, and R₆ and R₇ are hydrogen atoms. The fluorescentcompound represented by the general formula (8) is the fluorescentcompound of general formula (1) of when R₁, R₂, R₃, and R₄ are carboxylgroups.

EXAMPLE 1

The cis-fluorescent compound represented by the general formula (6) wasseparated and purified from a fluorescent compound containing thecis-fluorescent compound of general formula (6) and thetrans-fluorescent compound of general formula (7). Knownrecrystallization and column chromatography techniques were used for theseparation and purification of the cis-fluorescent compound. Thefluorescent compound was 95 mol % cis-fluorescent compound, and 5 mol %trans-fluorescent compound.

The fractions of the cis-fluorescent compound and the trans-fluorescentcompound were determined from the peak area ratio of high-performanceliquid chromatography (HPLC).

The fluorescent compound was dissolved in triethylene glycol dimethylether to prepare a 500 μM solution (freshness marker 1), and thefluorescence spectrum was measured. The fluorescence spectrum wasmeasured again after adding a 6 molar equivalent of spermidine to thefreshness marker 1. The fluorescence intensity a of the freshness marker1 after the addition of spermidine was 86 times higher than thefluorescence intensity b of the freshness marker 1 before addition ofspermidine.

EXAMPLE 2

The trans-fluorescent compound represented by the general formula (7)was separated and purified from a fluorescent compound containing thecis-fluorescent compound of general formula (6) and thetrans-fluorescent compound of general formula (7). Knownrecrystallization and column chromatography techniques were used for theseparation and purification of the trans-fluorescent compound. Thefluorescent compound was 15 mol % cis-fluorescent compound, and 85 mol %trans-fluorescent compound.

The fluorescent compound was dissolved in triethylene glycol dimethylether to prepare a 500 μM solution (freshness marker 1), and thefluorescence spectrum was measured. The fluorescence spectrum wasmeasured again after adding a 6 molar equivalent of spermidine to thefreshness marker 1. The fluorescence intensity a of the freshness marker1 after the addition of spermidine was 83 times higher than thefluorescence intensity b of the freshness marker 1 before addition ofspermidine.

EXAMPLE 3

The fluorescent compound containing two cis structures represented bythe general formula (8) was dissolved in triethylene glycol dimethylether to prepare a 500 μM solution (freshness marker 1), and thefluorescence spectrum was measured. The fluorescence spectrum wasmeasured again after adding a 2 molar equivalent of spermidine to thefreshness marker 1. The fluorescence intensity a of the freshness marker1 after the addition of spermidine was 26 times higher than thefluorescence intensity b of the freshness marker 1 before addition ofspermidine.

COMPARATIVE EXAMPLE 1

The fluorescent compound containing the cis-fluorescent compound ofgeneral formula (6) and the trans-fluorescent compound of generalformula (7) was dissolved in triethylene glycol dimethyl ether toprepare a 500 μM solution (freshness marker 1), and the fluorescencespectrum was measured. The fluorescent compound was 50 mol %cis-fluorescent compound, and 50 mol % trans-fluorescent compound. Thefluorescence spectrum was measured again after adding a 6 molarequivalent of spermidine to the freshness marker 1. The fluorescenceintensity a of the freshness marker 1 after the addition of spermidinewas 48 times higher than the fluorescence intensity b of the freshnessmarker 1 before addition of spermidine.

Table 1 shows the ratios of fluorescence intensities before and afteraddition of spermidine for the freshness markers 1 of Examples 1 to 3and Comparative Example 1. The fluorescence intensity ratio isrepresented as a ratio of fluorescence intensity a after the addition ofspermidine relative to the fluorescence intensity b before addition ofspermidine (a/b).

TABLE 1 Fluorescence intensity ratio Example 1 86 Example 2 83 Example 326 Comparative Example 1 48

FIG. 6 is a diagram representing the fluorescence intensities a of thefreshness markers 1 of Examples 1 to 3 and Comparative Example 1 afteraddition of spermidine. In FIG. 6, curve A represents the fluorescenceintensity a of the freshness marker 1 of Example 1 after addition ofspermidine, curve B represents the fluorescence intensity a of thefreshness marker 1 of Example 2 after addition of spermidine, curve Crepresents the fluorescence intensity a of the freshness marker 1 ofExample 3 after addition of spermidine, and curve D represents thefluorescence intensity a of the freshness marker 1 of ComparativeExample 1 after addition of spermidine.

The wavelength at which the highest fluorescence intensity occurs in thefluorescence spectrum differs for different fluorescent compoundstructures. The peak of the fluorescence spectrum for the freshnessmarker 1 of Comparative Example 1 occurs more toward the longerwavelength side as compared to the peak of the fluorescence spectrum forthe freshness markers 1 of Examples 1 and 2. This difference in the peakposition of the fluorescence spectrum is suggestive of formation of acis- and trans-fluorescent compound assembly in the freshness marker 1of Comparative Example 1.

As shown in Table 1 and FIG. 6, the freshness marker 1 of Example 1 hasa higher fluorescence intensity a than the freshness marker 1 ofComparative Example 1, and the ratio of fluorescence intensity arelative to the fluorescence intensity b is also greater. It cantherefore be said that the freshness marker 1 of Example 1 has higherspermidine sensitivity.

As shown in Table 1 and FIG. 6, the freshness marker 1 of Example 2 hasa smaller fluorescence intensity a than the freshness marker 1 ofComparative Example 1, but has a larger ratio of fluorescence intensitya relative to the fluorescence intensity b. It can be said from thisthat the freshness marker 1 of Example 2 has high spermidinesensitivity.

In the freshness marker 1 of Example 3, spermidine was added to thefreshness marker 1 in ⅓ of the amount added to the freshness markers 1of Examples 1 and 2 and Comparative Example 1. The ratio of fluorescenceintensity a relative to the fluorescence intensity b was 26, smallerthan that of the freshness marker 1 of Comparative Example 1. However, aratio of 70 or higher can be expected when the freshness marker 1 isequimolar. As shown in FIG. 6, the freshness marker 1 of Example 3 hasthe highest fluorescence intensity a compared to Examples 1 and 2 andComparative Example 1. It can be said from this that the freshnessmarker 1 of Example 3 has high spermidine sensitivity.

From these evaluation results, it can be said that the freshness markersusing fluorescent compounds containing a larger fraction ofcis-fluorescent compounds or trans-fluorescent compounds, and thefreshness marker using a fluorescent compound containing two cisstructures have high spermidine sensitivity.

The freshness markers 1 of Examples 1 and 2 and Comparative Example 1were used to produce freshness labels of Examples 4 and 5, andComparative Examples 2 and 3.

FIGS. 7A, 7B, and 7C are diagrams representing a fabrication method ofthe freshness label 100.

First, as shown in FIG. 7A, glass filters 102 were installed on a glasssheet 101, and the both ends of the glass filters 102 were fixed with anadhesive tape 103, as shown in FIG. 7B.

Thereafter, as shown in FIG. 7C, the freshness marker 1 was dropped witha pipette 104, and the glass filters 102 were impregnated with thefreshness marker 1 to fabricate the freshness label 100. Example 4 isthe freshness label 100 fabricated with the freshness marker 1 ofExample 1. Example 5 is the freshness label 100 fabricated with thefreshness marker 1 of Example 2. Comparative Examples 2 and 3 are thefreshness labels 100 fabricated with the freshness marker 1 ofComparative Example 1.

As illustrated in FIG. 8, the freshness label 100 fabricated in themanner described above was installed in a lidded bottle container Hcontaining a fresh food product P (e.g., kamaboko; minced and steamedfish meat). Here, the freshness label 100 was installed withoutcontacting the food product P. After installing the freshness label 100,the container was closed, and stored in a room temperature environment.The freshness label 100 was then observed for fluorescence state over atime period.

The fluorescence observation was conducted by observing a digital cameraimage of the fluorescence state after the freshness label 100 was takenout of the bottle container H, and irradiated with UV light in a roomwhere the influence of visible light was small.

FIG. 9 shows images of the freshness labels of Comparative Examples 2and 3, the freshness label of Example 4, and the freshness label ofExample 5 after 1, 2, 3, 4, and 5 days. A fluorescence intensity changewas visually observable by day 3 in the freshness label of Example 4,and by day 4 in the freshness label of Example 5, demonstrating that thefluorescence intensities of Examples 4 and 5 were higher than thefluorescence intensities of the freshness labels of Comparative Examples2 and 3.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the inventions. Indeed, the novel embodiments described hereinmay be embodied in a variety of other forms; furthermore, variousomissions, substitutions and changes in the form of the embodimentsdescribed herein may be made without departing from the spirit of theinventions. The accompanying claims and their equivalents are intendedto cover such forms or modifications as would fall within the scope andspirit of the inventions.

What is claimed is:
 1. A fluorescent compound mixture comprising: afirst fluorescent compound of a structure represented by the followinggeneral formula (1); and a second fluorescent compound of a structurerepresented by the following general formula (2), wherein the number ofmoles of the first fluorescent compound in the fluorescent compoundmixture is larger than the number of moles of the second fluorescentcompound in the fluorescent compound mixture, and the fluorescentcompound mixture has fluorescence characteristics that vary in thepresence of a subject substance, general formula (1) is:

wherein R₁ and R₂ each independently represent a carboxyl group and R₃and R₄ each independently represent a hydrogen atom, an alkyl group, analkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group, and general formula (2) is:

wherein R₅ and R₈ each independently represent a carboxyl group and R₆and R₇ each independently represent a hydrogen atom, an alkyl group, analkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group, and the second fluorescentcompound does not have the same structure as the first fluorescentcompound.
 2. The fluorescent compound mixture according to claim 1,wherein the subject substance is histamine.
 3. A freshness labelcomprising: a freshness marker comprising the fluorescent compoundmixture of claim 1; and a medium retaining the freshness marker.
 4. Asensing system comprising: a freshness marker comprising the fluorescentcompound mixture of claim 1; an ultraviolet source unit that emitsultraviolet light towards the freshness marker and an emission detectorthat senses the subject substance using an image pattern generated byexposing the freshness marker to ultraviolet light.
 5. The sensingsystem according to claim 4, wherein the emission detector determines aratio of luminescence intensities in the image pattern in the presenceand absence of the subject substance to sense the subject substance. 6.The sensing system according to claim 4, wherein the emission detectordetermines a difference between luminescence intensities in the imagepattern in the presence and absence of the subject substance to sensethe subject substance.
 7. A freshness marker for sensing a subjectsubstance, comprising: a fluorescent compound mixture that includes afirst fluorescent compound of a structure represented by the generalformula (1), and a second fluorescent compound of a structurerepresented by the following general formula (2); and a solvent in whichthe fluorescent compound mixture is dissolved, wherein the number ofmoles of the first fluorescent compound in the fluorescent compoundmixture is larger than the number of moles of the second fluorescentcompound in the fluorescent compound mixture, and the fluorescentcompound mixture has fluorescence characteristics that vary in thepresence of the subject substance, wherein general formula (1) is:

wherein R₁ and R₂ each independently represent a carboxyl group and R₃and R₄ each independently represent a hydrogen atom, an alkyl group, analkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group, and general formula (2) is:

wherein R₅ and R₈ each independently represents a carboxyl group and R₆and R₇ each independently represents a hydrogen atom, an alkyl group, analkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group, provided that the secondfluorescent compound does not have the same structure as the firstfluorescent compound.
 8. The freshness marker according to claim 7,wherein the number of moles of the first fluorescent compound in thefluorescent compound mixture is at least twice the number of moles ofthe second fluorescent compound in the fluorescent compound mixture. 9.The freshness marker according to claim 7, wherein the first fluorescentcompound and the second fluorescent compound form an assembly in thesolvent, and the number of moles of the first fluorescent compound inthe solvent and unassembled after the formation of the assembly is atleast twice the number of moles of the first fluorescent compound in theassembly.
 10. A sensing system comprising: the freshness marker of claim7; an ultraviolet source unit that emits ultraviolet light towards thefreshness marker; and an emission detector that senses the subjectsubstance using an image pattern generated by exposing the freshnessmarker to ultraviolet light.
 11. The sensing system according to claim10, wherein the emission detector determines a ratio of luminescenceintensities in the image pattern in the presence and absence of thesubject substance to sense the subject substance.
 12. The sensing systemaccording to claim 10, wherein the emission detector determines adifference between luminescence intensities in the image pattern in thepresence and absence of the subject substance to sense the subjectsubstance.
 13. A freshness label comprising: the freshness marker ofclaim 8; and a medium retaining the freshness marker.
 14. A sensingsystem comprising: the freshness marker of claim 8; an ultravioletsource unit that emits ultraviolet light towards the freshness marker;and an emission detector that senses the subject substance using theimage pattern generated by exposing the freshness marker to ultravioletlight.
 15. The sensing system according to claim 14, wherein theemission detector determines a ratio of luminescence intensities in theimage pattern in the presence and absence of the subject substance tosense the subject substance.
 16. The sensing system according to claim14, wherein the emission detector determines a difference betweenluminescence intensities in the image pattern in the presence andabsence of the subject substance to sense the subject substance.
 17. Afreshness label comprising: a freshness marker for sensing a subjectsubstance, comprising: a fluorescent compound mixture that includes afirst fluorescent compound of a structure represented by the generalformula (1), and a second fluorescent compound of a structurerepresented by the following general formula (2); and a solvent in whichthe fluorescent compound mixture is dissolved; and a medium retainingthe freshness marker, wherein the number of moles of the firstfluorescent compound in the fluorescent compound mixture is larger thanthe number of moles of the second fluorescent compound in thefluorescent compound mixture, and the fluorescent compound mixture hasfluorescence characteristics that vary in the presence of the subjectsubstance, wherein general formula (1) is:

wherein R₁ and R₂ each independently represent a carboxyl group and R₃and R₄ each independently represent a hydrogen atom, an alkyl group, analkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group, and general formula (2) is:

wherein R₅ and R₈ each independently represents a carboxyl group and R₆and R₇ each independently represents a hydrogen atom, an alkyl group, analkoxy group, a halogen atom, a hydroxyl group, a carboxyl group, or asubstituent containing a carboxyl group, provided that the secondfluorescent compound does not have the same structure as the firstfluorescent compound.
 18. The freshness label according to claim 17,wherein the number of moles of the first fluorescent compound in thefluorescent compound mixture is at least twice the number of moles ofthe second fluorescent compound in the fluorescent compound mixture.